Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Coffee aroma and flavor are key components in consumer preference for coffee varieties and brands. Coffee's characteristic aroma and flavor stems from a complex series of chemical reactions involving flavor precursors (Maillard reactions) that occur during the roasting of the bean. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, over 800 chemicals and biomolecules have been identified as contributing to coffee flavor and aroma. (Montavon et al., J. Agric. Food Chem., 51:2328-34 (2003)).
Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor in order to meet consumer preferences. Both aroma and flavor may be artificially imparted into coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (flavor). However, to date, there is little data concerning the influence of natural coffee grain components such as polysaccharides, proteins, and lipids on coffee aroma and flavor. One approach is to select varieties from the existing germplasm that have superior flavor characteristics. A disadvantage to this approach is that, frequently, the highest quality varieties also possess significant negative agronomics traits, such as poor yield and low resistance to diseases and environmental stresses. It is also possible to select new varieties from breeding trials in which varieties with different industrial and agronomic traits are crossed and their progeny are screened for both high quality and good agronomic performance. However, this latter approach is very time consuming, with one crossing experiment and selection over three growing seasons taking a minimum of 7-8 years. Thus, an alternative approach to enhancing coffee quality would be to use techniques of molecular biology to enhance those elements responsible for the flavor and aroma that are naturally found in the coffee bean, or to add aroma and flavor-enhancing elements that do not naturally occur in coffee beans. Genetic engineering is particularly suited to achieve these ends. For example, coffee proteins from different coffee species may be swapped. In the alternative, the expression of genes encoding naturally occurring coffee proteins that positively contribute to coffee flavor may be enhanced. Conversely, the expression of genes encoding naturally occurring coffee proteins that negatively contribute to coffee flavor may be suppressed. Another application of modern techniques is to use molecular information concerning the association of high quality with specific alleles to screen new varieties for the presence or absence of such using marker assisted breeding.
Coffees from different varieties and origins exhibit significant flavor and aroma quality variations when the green grain samples are roasted and processed in the same manner. The quality differences are a manifestation of chemical and physical variations within the grain samples that result mainly from differences in growing and processing conditions, and also from differences in the genetic background of both the maternal plant and the grain. At the level of chemical composition, at least part of the flavor quality can be associated with variations in the levels of small metabolites, such as sugars, acids, phenolics, and caffeine found associated with grain from different varieties. It is accepted that there are other less well characterized flavor and flavor-precursor molecules. In addition, it is likely that structural variations within the grain probably also contribute to differences in coffee quality. One approach to finding new components in the coffee grain linked to coffee quality is to study the genes and proteins differentially expressed during the maturation of grain samples in different varieties that possess different quality characteristics.
A group of proteins called the late embryogenesis abundant proteins (LEA), have been shown to accumulate in a coordinated fashion during the latter stages of cotton seed development (Dure, L, et al. Biochemistry 20: 4162-4178 (1981)). Dehydrin proteins (DHN) are a sub-group of the LEA proteins that have also been called the “LEA D-11 family” or LEA type 2 proteins (Close, T, Physiol. Plant 97: 795-803 (1996); Ingram, J, Annu. Rev. Plant Physiology Plant Mol Biol 47: 377-403 (1996)). Expression of the DHN proteins has been associated with the protection of various types of plant cells from osmotic stresses, such as those caused by desiccation, salt, and low temperature. (Skriver, K, et al. Plant Cell 2: 503-512 (1990); Allagulova, C R, et al. Biochemistry-Moscow 68: 945-951 (2003)).
In recent years, direct experimental evidence has linked increased expression of dehydrins with protection from osmotic stress. For example, Arabidopsis plants engineered to over-express a dehydrin fusion protein were found to have improved survival when exposed to low temperature (Puhakainen, T, et al. Plant Molecular Biology 54: 743-753 (2004)). Similarly, expression of a citrus dehydrin protein in transgenic tobacco has been shown to give increased tolerance to low temperature (Hara, M, et al. Planta 217: 290-298 (2003)). Other supporting evidence for the linkage of dehydrins and tolerance to low temperature induced stress are the observations that QTL loci for freezing tolerance and winterhardiness map very closely to dehydrins (Close T, 1996; Zhu, B, et al. Molecular and General Genetics 264: 145-153 (2000)). DHN genes are also expressed robustly in seeds toward the end of maturation, a period when the seed undergoes a developmentally programmed reduction in water content (Nylander, M, et al. Plant Molecular Biology 45: 263-279 (2001); Choi, D W, et al. Theoretical and Applied Genetics 100: 1274-1278 (2000)). The LEA/dehydrin proteins have been estimated to comprise up to 4% of the total seed protein, and are thought to be involved in protecting the embryo and/or other seed tissues from the osmotic stresses associated with the low water content of the mature seed (Roberts, J, et al. Plant Cell 5: 769-780 (1993); Wise, M, et al. Trends Plant Sci. 9: 13-17 (2004)).
Dehydrins are widely perceived to participate, with other LEA proteins, in the dehydration process that occurs during the late stages of seed maturation by assisting the acclimatization of seed tissues to the lower water content found in mature seeds (Close, Tm 1996); Nylander M, 2001). In addition, it is believed that the dehydrins synthesized seeds during maturation also continue to stabilize the associated cellular structures during seed quiescence. In this latter context, it has recently been proposed that dehydrins may also possess a radical-scavenging capability (Hara, M, 2003) and metal-binding properties (Alsheikh, M K, et al. J. Biol. Chem. 278: 40882-40889 (2003)), both characteristics that are likely to be useful during long periods of seed storage.
A considerable number of dehydrin proteins have been isolated and studied, and the precise physiochemical and/or structural mechanism(s) whereby these proteins function to protect cells from osmotic stress in-vivo is under investigation. The dehydrins are very hydrophilic proteins and exhibit an unusually low level of recognizable structure (Close T, 1996; Soulages, J L, et al. Plant Physiology 131: 963-975 (2003)). A key element of the dehydrins is believed to be the presence of one or more 15 amino acid, lysine rich, stretches called the “K motifs,” which are predicted to form class A amphipathic alpha-helices (Close, T, 1996; Close, T J Physiol. Plant 100: 291-296 (1997)). Dehydrins can also contain two other motifs, an N-terminal “Y segment” (consensus V/TDE/QYGNP) and a serine rich “S segment,” the latter of which can be phosphorylated and is thought to participate in nuclear localization (Close T J, 1997; Godoy, J A, et al. Plant Mol. Biol. 1921-1934 (1994)). It has been proposed that the short amphipathic K segments of dehydrin polypeptides functionally interact with the solvent-exposed hydrophobic patches of proteins that are undergoing partial denaturation, and thereby block protein aggregate formation (Close T, 1996). Amphipathic K helixes may also be involved in binding membrane lipids, and thus could play a more specific role in protecting lipoproteins, proteins located in membranes, and/or membrane structure itself (Close T, 1996; Koag, M C, et al. Plant Physiology 131: 309-316 (2003)). An alternative proposal for at least part of the protective effect of dehydrins is the ability of these very stable, but relatively unstructured, proteins to tightly bind and organize water molecules (Soulages J L, 2003). This latter effect could lead to reduced water loss from cells, and could also improve the stability of certain macromolecules by the development of dehydrin based region of more tightly bound “ordered” water around these molecules.
Despite the involvement of dehydrin proteins in plant resistance to osmotic stresses such as drought and salt stress, and the probable importance of the dehydrins during grain development, little information is available on these genes in coffee. In coffee, little is understood about the number of dehydrins, their protein structure, their expression levels and distribution in different tissues of the coffee plant and among coffee species, as well as during coffee grain and pericarp maturation, and the regulation of their expression on the molecular level. Thus, there is a need to identify, isolate and characterize coffee dehydrin proteins, genes, and genetic regulatory elements. Such information will enable coffee dehydrin proteins to be genetically manipulated, with the goal of improving the aroma and flavor of the coffee, as well as imparting other phenotypic advantages associated with improved osmotic stress resistance.
Dehydrins, which are expressed at relatively high levels at the end of grain maturation, are of interest because of the potentially important roles they have in organizing water molecules in the coffee grain and in stabilizing macromolecules and organelles within the mature dehydrated grain. At least part of this protective effect is believed to be due to the ability of the dehydrins and other LEA proteins to stabilize different water/protein/lipid interfaces. Because water levels can influence the spectrum of products formed in the Maillard reaction (Turner, J, et al. J. Agric. Food Chem 50: 5400-5404 (2002)), the availability and organization of water molecules in the coffee grain may influence the flavor generating Maillard reaction occurring during the roasting of coffee.